Post-translational modification and calcium binding are key pre-requisites for centrosome duplication and separation. These processes are regulated in healthy tissues and are defective in disease states such as cancer. Abnormalities, such as amplified and multiple centrosomes, are often observed in human breast, colorectal, and other cancers. Centrin is an EF-hand protein that plays both structural and regulatory roles in the centrosome. This calcium-binding protein interacts at low calcium levels with a novel 1242-amino acid protein known as Sfi1, which contains up to 23 centrin-binding sites. Coupled biophysical, structural, and dynamic analyses of the centrin/SfM complex are essential to the understanding of its biological function. In Aim I, we will determine the interactions involved in the formation of the centrin-Sfil complex and observe how they change during the dynamic contraction and elongation of Sfi1. Our experiments will address key questions underlying the molecular basis of this complex interaction. We will study the processes of calcium-binding and phosphorylation in centrin and relate them to the structural changes that trigger contraction and elongation in Sfi1. In Aim 2, we will determine the structure of Sfi1 and measure the kinetics of its contraction and elongation. We expect that structural characterizations of Sfi1 will yield insight into the structure-function relationship of this protein. Our results will elucidate how newly duplicated centrosomes separate and migrate to opposite sides of the nucleus. With the completion of the proposed project, we expect to attain our short-term goal of describing at the molecular level the conformational changes that occur in centrin and Sfi1 upon complex formation. We hope to define the molecular mechanism of Sfi1 contraction and the conditions that facilitate this event. In addition, characterization of the phosphorylated form of centrin will contribute greatly to structural knowledge of this post-translational modification, since few phosphorylated proteins have been structurally resolved to date. The significance of these results lies in our understanding of the regulatory and structural aspects of cell division at the centrosome level. Our findings will provide essential information on the mechanisms by which centrosomal proteins function and help define their biological activities.